The function of miR165/166 in plant growth and development has been extensively studied, however, its roles in abiotic stress responses remain largely unknown. Here, we report that reduction in the expression of miR165/166 conferred a drought and cold resistance phenotype and hypersensitivity to ABA during seed germination and post-germination seedling development. We further show that the ABA hypersensitive phenotype is associated with a changed transcript abundance of ABA-responsive genes and a higher expression level of ABI4, which can be directly regulated by a miR165/166 target. Additionally, we found that reduction in miR165/166 expression leads to elevated ABA levels, which occurs at least partially through the increased expression of BG1, a gene that is directly regulated by a miR165/166 target. Taken together, our results uncover a novel role for miR165/166 in the regulation of ABA and abiotic stress responses and control of ABA homeostasis. Functions of miRNAs in plant development and stress responses have been extensively studied. However, little is known about how a miRNA may perform critical functions in both plant development and abiotic stress responses. One well-known miRNA, miR165/166, has critical roles in plant development. In this study, we show that this miRNA also has important functions in ABA and abiotic stress responses. Since the expression level of miR165/166 can be reduced to different extents using short tandem target mimicry (STTM), in the present work, we used STTM165/166 transformants with moderate developmental phenotype to examine its potential role in abiotic stress responses. Our results show that miR165/166 plays critical roles in drought and cold stress resistance as well as in ABA responses. Our work reveals that miR165/166-mediated regulatory module is linked with ABA responses and homeostasis through ABI4 and BG1.

MicroRNAs (miRNAs) regulate gene expression and play critical roles in growth and development as well as stress responses in eukaryotes. miRNA biogenesis in plants requires a processing complex that consists of the core components DICER-LIKE 1 (DCL1), SERRATE (SE) and HYPONASTIC LEAVES (HYL1). Here we show that inactivation of functionally redundant members of the SnRK2 kinases, which are the core components of abscisic acid (ABA) and osmotic stress signaling pathways, leads to reduction in miRNA accumulation under stress conditions. Further analysis revealed that the steady state level of HYL1 protein in plants under osmotic stress is dependent on the SnRK2 kinases. Additionally, our results suggest that the SnRK2 kinases physically associate with the miRNA processing components SE and HYL1 and can phosphorylate these proteins in vitro. These findings reveal an important role for the SnRK2 kinases in the regulation of miRNA accumulation and establish a mechanism by which ABA and osmotic stress signaling is linked to miRNA biogenesis. miRNAs play critical roles in various biological processes. miRNA biogenesis in both animals and plants are generated by a miRNA processing complex. Studies on specific miRNA function in different biological process have been extensively reported. However, how miRNA biogenesis is modulated, especially by environmental stimuli is poorly understood. The family of 10 redundant members of SnRK2 protein kinases are the central components of abscisic acid (ABA) and osmotic stress signaling pathways. In this study, our work reveals a novel role for SnRK2s in the modulation of miRNA levels. Importantly, we show that the SnRK2 kinases are required for the maintenance of the steady state level of HYL1 protein and the miRNA processing complex components, SE and HYL1, are potential substrates of the SnRK2 kinases. Our study provides a new mechanism of regulation of miRNA biogenesis and paves the way for further understanding of the impact of ABA and osmotic stress signaling on miRNA production.

Heat stress (HS) adversely affects plant growth and productivity. The class A1 heat stress transcription factors (HSFA1s) act as master regulators in the plant response to HS. However, how HSFA1-mediated transcriptional reprogramming is modulated during HS remains to be elucidated. Here, we report that a module formed by the microRNAs miR165 and miR166 and their target transcript, PHABULOSA (PHB), regulates HSFA1 at the transcriptional and translational levels to control plant HS responses. HS-triggered induction of MIR165/166 in Arabidopsis thaliana led to decreased expression of target genes including PHB. MIR165/166 overexpression lines and mutations in miR165/166 target genes enhanced HS tolerance, whereas miR165/166 knockdown lines and plants expressing a miR165/166-resistant form of PHB were sensitive to HS. PHB directly repressed the transcription of HSFA1s and globally modulated the expression of heat stress–responsive genes. PHB and HSFA1s share a common target gene, HSFA2, which is essential for activation of plant responses to HS. PHB physically interacted with HSFA1s and exerted an antagonistic effect on HSFA1 transcriptional activity. PHB and HSFA1s co-regulated transcriptome reprogramming upon HS. Together, these findings indicate that heat-triggered regulation of the miR165/166–PHB module controls HSFA1-mediated transcriptional reprogramming and plays a critical role during HS in Arabidopsis.

Plants have evolved a complex regulatory network to cope with heat stress (HS), which includes microRNAs (miRNAs). However, the roles of the entire miRNA biogenesis machinery in HS responses remain unclear. Here, we show that HS induces the majority of miRNAs primarily through the enhanced nuclear localization of HYPONASTIC LEAVES 1 (HYL1), rather than by upregulating MIR gene transcription in Arabidopsis (Arabidopsis thaliana). Disruption of miRNA biogenesis increases plant susceptibility to HS. We also demonstrate that HYL1 phosphorylation modulates its nuclear localization during HS, which is critical for miRNA induction and thermotolerance. MAP KINASE3 (MPK3) phosphorylates and stabilizes the phosphatase C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1 (CPL1), while CPL1 inhibits MPK3 activity, creating a feedback loop that regulates HYL1 phosphorylation. Disruption of MPK3 function results in increased nuclear HYL1 levels and miRNA production, conferring enhanced HS tolerance to mpk3 mutants. These findings reveal a mechanism by which plants enhance miRNA biogenesis during HS, offering insights into the regulatory networks that govern plant thermotolerance.